DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s arguments and amendments have been thoroughly reviewed and considered. Claims 22-24, 41-46, and 56 have been canceled. Claims 1-2, 7, 9, 12, 14, 16-17, 21, and 62 -73 are pending and are examined on the merits herein.
Response to Applicant’s Amendments
Nucleotide and/or Amino Acid Sequence Disclosures
The drawings were objected to because Figures 7 and 8 contained sequences without sequence identifiers. In light of Applicant’s amendments to the drawings and specification submitted 5/6/2026, these objections have been withdrawn. Additionally, in the Non-Final Rejection mailed 3/4/2026, it was also stated that Applicant is required to abide by the sequence compliance rules under ST.26, and not ST.25. Applicant has provided arguments and Remarks against this point (Remarks, page 10). Applicant’s arguments are persuasive, and so the current Sequence Listing in ST.25 format has been fully considered by the Examiner.
Drawing Objections
The drawings were objected to because Figures 5, 9-10C, 12, 13A, 13B, 13D, 13E, 13G, and 14 were not legible. In light of Applicant’s amendments to the drawings submitted 5/6/2026, these objections have been withdrawn.
Claim Objections
Claims 1-2, 7, 17, 21 62, and 71-73 were objected to due to various informalities. In light of Applicant’s amendments to the claims submitted 5/6/2026, these objections have been withdrawn for claims 1-2, 7, 21, 62, and 71-73. The amendments to claim 17 do not fully address the objection made, and so this objection has been maintained. See also new grounds of objection below.
35 USC 112(b) Rejections
Claims 1-2, 7, 9, 12, 14, 16-17, 21, and 62-73 were rejected due to various indefiniteness issues. In light of Applicant’s amendments to the claims submitted 5/6/2026, the rejection for claim 12 has been withdrawn. With regard to the other rejections, Applicant’s arguments and amendments have been thoroughly reviewed and considered. These rejections have been maintained. See “Response to Applicant’s Arguments” below.
35 USC 103 Rejections
Claims 1-2, 7, 9, 12, 14, 16, 21, 64-67, 69, and 70 were rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS) in view of Lee (Dissertation, 2020).
Claims 17 and 72 were rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS), in view of Lee (Dissertation, 2020), and further in view of Takara (2019).
Claim 63 was rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS), in view of Lee (Dissertation, 2020), and further in view of Liu et al. (Front. Microbiol., 2019).
Claim 68 was rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS), in view of Lee (Dissertation, 2020), and further in view of Kim et al. (Cell, 2020).
Claim 71 was rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS), in view of Lee (Dissertation, 2020), and further in view of Bibillo et al. (US 2019/0071711 A1).
Claim 73 was rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS), in view of Lee (Dissertation, 2020), and further in view of Parker et al. (Journal of Applied Microbiology, 2010).
Applicant’s arguments and amendments have been thoroughly reviewed and considered. In light of Applicant’s amendments to the claims submitted 5/6/2026, these rejections have been withdrawn, but see new grounds of rejection and “Response to Applicant’s Arguments” below.
Response to Applicant’s Arguments
Regarding the 35 USC 112(b) Rejections, Applicant’s Remarks concerning the pathogen-specific oligonucleotide primer do not appear to address the thrust of the rejection. This rejection was based on the “3’ end” language used for multiple components within the primer sequence (see para. 18 of the Non-Final Rejection), and was not based on the pathogen specific consensus sequence. Applicant has not amended the claim to address these “3’ end” recitations, nor has Applicant provided additional clarity related to this point, and so this rejection has been maintained.
In para. 19 of the Non-Final Rejection, the language used to describe the adapters of claim 1 was rejected as being indefinite. Applicant does not appear to address this rejection at all in their Remarks, nor has Applicant amended the adapter language associated with claim 1, and so this rejection has also been maintained.
Related to the use of the term “cDNA splint” in claims 1-2 and 62, this term was rejected because it was unclear if the sequence of the cDNA splint must be RNA or cDNA. Applicant’s arguments do not provide any clarity to this point, nor do the citations to portions of the instant specification (page 13 of Applicant’s Remarks). Thus, this rejection has been maintained. Applicant also does not address in any fashion the similar rejections associated with “cDNA UMI,” for claims 1-2 and 62 and so these rejections have also been maintained.
Applicant also does not address the rejections associated with para. 21 of the Non-Final Rejection in their Remarks, and so this rejection has been maintained.
Regarding the rejections to claims 17 and 71-73, these claims were not rejected because it was unclear what the terms “single-pot,” “split-pot,” “open-tube,” “closed-tube,” and “multi-tubed” meant, but it was unclear how these various chemistries were incorporated into the method described by claim 1. Though Applicant refers to their specific working examples as examples of these various types of chemistries, these examples are much more detailed than the instant claims, for example in terms of the required reagents and reaction conditions. MPEP 2111.01 II notes that it is improper to import claim limitations from the specification, and so these examples alone do not alleviate the indefiniteness issues associated with the claims. As Applicant has not amended the claims to address the thrust of the rejection, nor do Applicant’s Remarks clarify the associated indefiniteness issues, these rejections are maintained.
Applicant also does not address the rejections associated with paras. 25-26 (associated with claim 62) of the Non-Final Rejection in their Remarks, and so these rejections have been maintained.
Regarding the 35 USC 103 Rejections, Applicant argues that the cited references do
not teach the claimed limitations, and that the obviousness rejections rely on impermissible hindsight (Remarks, page 16). Specifically, Applicant argues that the references fail to teach the newly amended portion of claim 1 (Remarks, page 17). On page 18 of their Remarks, Applicant argues that Chappleboim teaches away from the claimed invention. On page 19 of their Remarks, Applicant argues that Lee is not analogous art, and so cannot be used in an obviousness rejection. On page 20 of their Remarks, Applicant argues that there is a lack of motivation to combine Chappleboim in view of Lee as is done in the obviousness rejections, and so this rejection relies on impermissible hindsight. Page 21 of Applicant’s Remarks reiterates that Chappleboim in view of Lee does not teach two distinct unique molecular identifiers as is done in the instant claims. Applicant also states that the Examiner improperly relies on “natural result” reasoning and inherency. On pages 23-24 of their Remarks, Applicant states that the secondary references do not cure the deficiencies of Chappleboim in view of Lee. On pages 24-26 of Applicant’s Remarks, allegedly unexpected results of the claimed invention are discussed.
Regarding Applicant’s amendments to the claims, and specifically those of instant claim 1, Chappleboim in view of Lee teaches that the anchoring oligonucleotide and the pathogen specific oligonucleotide primer are separate oligonucleotide sequences (see paras. 46 and 48 of the Non-Final Rejection). However, the pathogen specific oligonucleotide primer of Chappleboim in view of Lee is not hybridized to the nucleic acid before reverse transcription, nor are the UMI sequences on the two oligonucleotides different. Thus, the Examiner finds this argument persuasive, and this is the basis for the new grounds of rejection below.
Regarding teaching away, MPEP 2141.02 VI states, “"the prior art’s mere disclosure of more than one alternative does not constitute a teaching away from any of these alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed…." In re Fulton, 391 F.3d 1195, 1201, 73 USPQ2d 1141, 1146 (Fed. Cir. 2004),” and MPEP 2143.01 V states, ‘[a] given course of action often has simultaneous advantages and disadvantages, and this does not necessarily obviate motivation to combine’"(quoting Medichem, S.A. v. Rolabo, S.L., 437 F.3d 1157, 1165, 77 USPQ2d 1865, 1870 (Fed. Cir. 2006).” Applicant does not specifically quote Chappleboim for evidence of teaching away, and simply states that Chappleboim “repeatedly emphasizes the advantages of consolidating multiple functionalities into a single reverse transcription primer.” However, at no point does Chappleboim specifically disparage the use of multiple, structurally distinct oligonucleotides, and so cannot be said to teach away from the claimed invention. Though the invention of Chappleboim alone may not have a group of oligonucleotides with the same exact structure as those of the claimed invention, and the structure of the oligonucleotides of Chappleboim may come with their own distinct advantages, the presence of these advantages alone does not obviate the motivation to combine Chappleboim with Lee as is done in the Non-Final Rejection.
Thus, this argument is not considered persuasive.
Regarding analogous art, MPEP 2141.01(a) I states that, “A reference is analogous art to the claimed invention if: (1) the reference is from the same field of endeavor as the claimed invention (even if it addresses a different problem); or (2) the reference is reasonably pertinent to the problem faced by the inventor (even if it is not in the same field of endeavor as the claimed invention).” In determining if a reference is in the same field of endeavor as the claimed invention, “These circumstances are to be weighed "from the vantage point of the common sense likely to be exerted by one of ordinary skill in the art in assessing the scope of the endeavor." Airbus, 41 F.3d at 1380. See also Donner Technology, LLC v. Pro Stage Gear, LLC, 979 F.3d 1353, 2020 USPQ2d 11335 (Fed. Cir. 2020); Sanofi-Aventis, 66 F.4th at 1378; and Netflix, Inc. v. DivX, LLC, 80 F.4th 1352, 1358-59, 2023 USPQ2d 1057 (Fed. Cir. 2023) ("The field of endeavor is ‘not limited to the specific point of novelty, the narrowest possible conception of the field, or the particular focus within a given field.’") (quoting Unwired Planet, LLC v. Google Inc., 841 F.3d 995, 1001, 120 USPQ2d 1593, 1597 (Fed. Cir. 2016)).” Thus, a reference does not have to provide teachings as narrow as the claimed invention. Though Lee does provide methods related to hydrogels, the reference also teaches workflows for sequencing library preparation (see section 4.5 starting on page 56 of Lee). As the claimed invention is also focused on sequencing library preparation, both Lee and the claimed invention are considered to be in the same field of endeavor, and Lee is therefore able to be used in obviousness rejections.
Thus, this argument is not considered persuasive.
Regarding impermissible hindsight, MPEP 2145 X (A) states, “[a]ny judgment on obviousness is in a sense necessarily a reconstruction based on hindsight reasoning, but so long as it takes into account only knowledge which was within the level of ordinary skill in the art at the time the claimed invention was made and does not include knowledge gleaned only from applicant’s disclosure, such a reconstruction is proper." In re McLaughlin, 443 F.2d 1392, 1395, 170 USPQ 209, 212 (CCPA 1971).” In the combination of Chappleboim in view of Lee, Applicant’s instant specification is not recited in the rationale for combining these references, and so said combination is not considered to rely on impermissible hindsight.
In combining Chappleboim in view of Lee, para. 45 of the Non-Final Rejection explains similarities in the teachings of the two references that would motivate the ordinary artisan to combine said references – namely, that both are drawn to similar sequencing methods, and that Lee teaches general mRNA library preparation guidance – and provides evidence that the combination of references would have predictable results (see MPEP 2143 I (A), which states, “The rationale to support a conclusion that the claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination yielded nothing more than predictable results to one of ordinary skill in the art.”). In para. 46, the teachings of Lee are used to arrive at the anchoring oligonucleotide, with the rationale that “By combining the capturing and reverse transcription steps, this would cut down on required time and resources to perform the overall method, as separate anchoring sequences and RT-primers would not need to be designed. Lee teaches that this type of oligonucleotide can specifically be used as a RT primer (page 10, para. 1) providing a reasonable expectation of success.” In para. 50, the structure of the sequencing adapters of Lee are used in Chappleboim, with the rationale that, “the sequencing adapters of the references provide the same function (labeling the amplification products so they may be sequenced and more easily analyzed), and are added the same way. The use of the sequencing adapter designs of Lee in Chappleboim would therefore amount to simple substitution. MPEP 2143 I (B) states, “The rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.” As both adapter designs are known in the art, and their use is made clear by both Chappleboim and Lee, such substitution would be prima facie obvious.” Thus, each aspect of the combination of Chappleboim and Lee was accompanied by a rationale and a reasonable expectation of success. Note that in the 35 USC 103 Rejections below, some of this rationale is altered due to the new grounds of rejection required, but the general motivation for combining the two references, and the presence of rationale and a reasonable expectation of success for each presented obviousness rejection, remains.
As noted above, particular advantages and disadvantages associated with a particular combination of references do not obviate a finding of obviousness. Additionally, in combining references, MPEP 2144 I states, "The rationale to modify or combine the prior art does not have to be expressly stated in the prior art; the rationale may be expressly or impliedly contained in the prior art or it may be reasoned from knowledge generally available to one of ordinary skill in the art, established scientific principles, or legal precedent established by prior case law," MPEP 2141.03 I states, "'A person of ordinary skill in the art is also a person of ordinary creativity, not an automaton.' KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 421, 82 USPQ2d 1385, 1397 (2007). "[I]n many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle." Id. at 420, 82 USPQ2d 1397. Office personnel may also take into account "the inferences and creative steps that a person of ordinary skill in the art would employ." Id. at 418, 82 USPQ2d at 1396," and MPEP 2144 II states, "'Indeed, we have repeatedly held that an implicit motivation to combine exists not only when a suggestion may be gleaned from the prior art as a whole, but when the ‘improvement’ is technology-independent and the combination of references results in a product or process that is more desirable, for example because it is stronger, cheaper, cleaner, faster, lighter, smaller, more durable, or more efficient. Because the desire to enhance commercial opportunities by improving a product or process is universal—and even common-sensical—we have held that there exists in these situations a motivation to combine prior art references even absent any hint of suggestion in the references themselves.'"
Taken together, this means that neither Chappleboim nor Lee need to provide an explicit motivation to alter their teachings in order to be used in an obviousness rejection. Furthermore, in making an obviousness rejection, rationale that would be generally available to the ordinary artisan may be used, and this rationale can be based on the ordinary artisan’s skill, knowledge, and creativity in the art. The Office then distinctly points out that such non-explicit rationales can be those which enhance efficiency, which would be accomplished by the combination of Chappleboim in view of Lee above, particularly with regard to the anchoring oligonucleotide combination. Thus, it is the Examiner’s position that the full combination of Chappleboim in view of Lee presented in the Non-Final Rejection relied on rationale that results from similarities between the references (such as those of MPEP 2143 I (A) and (B)) and rationale which, while not explicit in the references, would be capable of being reached by the ordinary artisan.
Thus, this argument is not persuasive.
Regarding the claimed dual-UMI structure of the claimed invention, as noted above, the Examiner agrees that Chappleboim in view of Lee does not teach this feature. This argument is thus persuasive and is the reason for the new grounds of rejection provided below.
Regarding the “natural result” reasoning described by Applicant, Applicant does not specifically point out where the Examiner uses such reasoning. It is believed that Applicant is referring to instant such as those of para. 48 of the Non-Final Rejection, where the Examiner states, “Chappleboim teaches that during PCR, the forward primer hybridizes to the RT-primer region via the PCR handle, and upon extension, would then contain the UMI sequence and the pathogen specific consensus sequence. The minimal and extended cDNA splints would also naturally exist on this primer before and after extension…” As also stated in this paragraph, this reasoning is based on the 35 USC 112(b) Rejections and the Claim Interpretations used by the Examiner. The 35 USC 112(b) Rejections regarding particular terms related to the oligonucleotides in the claims have been maintained. In replying to many of these rejections, Applicant merely points to where these terms appear in the instant specification. However, as noted above, it is improper to import claim limitations from the specification, and for these terms, Applicant has not provided clear, explicit definitions. It is generally unclear how all of Applicant’s claimed features could exist without using the interpretations provided by the Examiner. For example, for the pathogen specific oligonucleotide primer, the “extended 3’ end cDNA splint” cannot exist before extension. Given this fact and the indefiniteness rejections, the claim interpretations of the terms in instant claim 1 stand, and therefore, the “natural result” reasoning does as well.
Thus, this argument is not persuasive.
Regarding the arguments for the secondary references, Applicant’s arguments generally do not address the thrust of each of the references, and attack each of the secondary references individually. In response to Applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Each of the obviousness rejections relying on secondary references contains a rationale and reasonable expectation of success, and so these are considered proper. In combining Parker with Chappleboim in view of Lee, Applicant states that MDA has known disadvantages that would contrary to Chappleboim’s design. Applicant is directed to the previous arguments concerning advantages and disadvantages associated with combinations of references, and how the presence of disadvantages alone does not obviate an obviousness rejection.
Thus, these arguments are not considered persuasive.
Regarding unexpected results, MPEP 716.02 discusses the considerations for such allegations. These considerations require Applicant to establish that results are in fact unexpected and significant, including explaining any associated data, to compare the produced results with those of the closest prior art, and to describe unexpected results that are commensurate in scope with the claimed invention.
Applicant points to their LeaSH protocol, which is the focus of their working examples. The results in Figures 6, 9, and 11-15 are discussed. Regarding Figure 6, Applicant states that the LeaSH method with Illumina sequencing shows that the claimed oligonucleotides “preserve barcode fidelity and quantitative molecular tracking” and the ability to quantify multiple transcript abundances from low-RNA input samples. Regarding Figure 9, Applicant focuses on how the shown LeaSH results show multiplex detection with very low provided amounts of starting RNA. In both of these examples, it is not clear why such results are superior or unexpected relative to those produced by other methods in the prior art. For instance, Chappleboim shows that their methods have a low limit of detection (see the LoD of Figure 4C) which can be detected in both single and pooled samples, and can act on multiple targets (see Figure 5). For Figures 11-14, Applicant is focused on how the library preparation oligonucleotides of the claimed invention can operate with many different workflows while maintaining functionality, and states that this would not have been predictable. However, many oligonucleotides can operate under many different specific protocols, such as amplification primers or probes, so it unclear why the results shown by Applicant for their library preparation oligonucleotides would not be predictable. For Figures 14-15, specific results are not discussed in the context of how the results are particularly significant or unexpected. Here, the results appear to be focused on successful detection of SARS-CoV-2, but such detection can be done via other means, such as those of Chappleboim. Applicant states that these results produced are unexpected over the prior art, and particularly in view of Chappleboim, and that the results produced would not have been reasonably predicted, but does not explain why this would be the case. In addition, Applicant presents earlier in their arguments that the method of Chappleboim is entirely different than that of the claimed invention, and allegedly teaches away from the claimed invention, so it is not entirely clear why Applicant then chose this reference to be the closest prior art to the claimed invention.
The results shown in these figures pointed to by Applicant also are not commensurate in scope with the claimed invention. For example, the working examples of the instant specification use particular pathogens, and thus particular consensus sequences, use particular arrays, and have particular size selection processes and chemistries that are not encompassed by instant claim 1.
Thus, this argument is not considered persuasive.
Overall, most of Applicant’s arguments were not found to be persuasive. However, Applicant’s claim amendments require new grounds of rejection, which are provided below. It is noted that portions of Chappleboim in view of Lee as they relate to the newly amended claims are reiterated below.
Claim Objections
Claim 1 is objected to because of the following informalities: in the newly amended wherein clause paragraph, in line 1, “pathogen-specific” should read “pathogen specific,” and it is recommended that the phrase “unique molecular identifier” in line 4 of the phrase be abbreviated to “UMI.” Appropriate correction is required.
Claim 17 is objected to because of the following informalities: for each of the presented options, it is recommended to state one of “with single-pot, closed tube chemistry,” “with single-pot, open tube chemistry,” and “with single-pot, multi-tube chemistry using…”. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-2, 7, 9, 12, 14, 16-17, 21, and 62-73 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 is rejected because the pathogen-specific oligonucleotide primer is unclear. Specifically, the primer requires a minimal and extended 3’ end cDNA splint, as well as a 3’ end cDNA UMI. It is unknown how all of these components can simultaneously exist at the 3’ end of a single sequence. These limitations for this oligonucleotide will be interpreted as though they are referring to the 3’ ends of each individual portion (i.e. the 3’ end of the UMI sequence and the 3’ end of the splint sequences).
Similarly, the adapters of claim 1 are unclear as they require multiple simultaneous 3’ or 5’ components. It is unknown how all of these components can simultaneously exist at the 3’ or 5’ end of a single sequence. These limitations for the adapter oligonucleotides will be interpreted as though they are referring to the 3’ or 5’ ends of each individual portion of the adapter, similar to the interpretation above for the pathogen-specific oligonucleotide primer.
Claims 1-2 and 62 are rejected due to the use of the phrases “cDNA splint” and “cDNA UMI.” Specifically, as cDNA is typically made from reverse transcription of RNA, it is unclear if these terms indicate that the splint and UMI sections must be made of cDNA, or if they are simply used on oligonucleotide sequences that will be extended to include cDNA. Particularly, as cDNA generally contains exon sequences of genomic DNA, and a UMI is defined in the instant specification as “an oligonucleotide sequence that can be used to identify an individual molecule or nucleic acid fragment present in the sample” (see para. 101), it is unclear how such a sequence would be comprised of cDNA. Therefore, these terms will be interpreted to indicate that the oligonucleotides they are a part of should be composed of DNA, and that these oligonucleotides should at least partially form cDNA when extended.
Claim 2 is also rejected for reasons similar to those of claim 1. Specifically, for oligonucleotides (vii) and (viii), multiple simultaneous 5’ or 3’ components are recited. It is unknown how all of these components can simultaneously exist at the 3’ or 5’ end of a single sequence. These limitations for oligonucleotides (vii) and (viii) will be interpreted as though they are referring to the 3’ or 5’ ends of each individual portion, similar to the interpretation above for the pathogen-specific oligonucleotide primer of instant claim 1.
Claims 2, 7, 9, 12, 14, 16-17, 21, and 62-73 are rejected due to their dependence on rejected claim 1.
Claims 17 and 71-73 all recite that the method of claim 1 must be performed with particular types of chemistries, without specifying that these chemistries apply to a particular step of the method. Thus, the recited chemistries are considered to apply to the entirety of the method of claim 1. However, the entirety of the method of claim 1 involves steps which cannot be performed in a tube, such as obtaining a sample from the subject. Additionally regarding claim 17, claim 1 recites the use of sequencing, which could not be performed if closed tube chemistry was used. Thus, the metes and bounds of the claims are unclear. It will be interpreted as though the chemistries recited in these claims are specifically referring to the library preparation step of claim 1.
Claim 62 is rejected because the preamble recites “preparing the library comprises further nucleic acid amplification,” when initial amplification is not explicitly recited in claim 1. It is therefore unclear where initial amplification during the library preparation is meant to occur relative to this further amplification. Prior art will be considered to read on this claim if it teaches the stated oligonucleotides within the context of library preparation generally.
Additionally, claim 62 is rejected because for oligonucleotide (viii), multiple simultaneous 5’ components are recited. It is unknown how all of these components can simultaneously exist at the 5’ end of a single sequence. These limitations for oligonucleotides (viii) will be interpreted as though they are referring to the 5’ ends of each individual portion, similar to the interpretation above for the pathogen-specific oligonucleotide primer of instant claim 1.
Claim Interpretation
For clarity, the interpretation of the required oligonucleotides of the instant claims is provided below. It is noted that the function of each individual oligonucleotide during library preparation is not specifically stated in the claims. For example, the particular uses of the primers, the anchored oligonucleotide, adapters, blocking oligonucleotide, and template switching oligonucleotides in the library preparation process of the claims are not stated. Thus, prior art will considered to read on the claimed oligonucleotides if it teaches use of oligonucleotides with the claimed required structural components generally. It is noted that during library preparation, reverse transcription must occur, where the anchored oligonucleotide and pathogen specific oligonucleotide primer must be hybridized to the target nucleic acid before said reverse transcription commences. This limitation must also be present in the prior art.
The term “splint” has no specific definition in the instant specification. Based on its use in the claims, a “splint” sequence will be interpreted as a sequence capable of hybridizing to another sequence.
In claim 1:
Step (a) recites the use of a sample and “extracting nucleic acids” from said sample. However, “extracting” has no specific definition in the instant specification, and so will be interpreted to encompass any method that allows nucleic acids within the sample to be directly interacted with/manipulated. This would involve methods that include cell lysis to access nucleic acids, or methods involving the use of particular buffers that allow for access to nucleic acids.
For the anchored oligonucleotide, the term “anchored” has no specific definition in the instant specification. Para. 9 describes amplification as being “anchored” to consensus sequences, and the methods below para. 120 recite anchored multiplexing in a well plate, where it appears that anchoring involves attachment to the well plate. In considering the broadest reasonable interpretation of the term in light of the prior art, an “anchored oligonucleotide” will be considered one that is capable of being anchored to a substrate, without specifically requiring that the oligonucleotide be anchored to said substrate in the method of claim 1.
For the pathogen-specific oligonucleotide primer, a pathogen specific consensus sequence is defined in the instant specification in para. 103 as a conserved region in a pathogen’s genome that is well-conserved across a plurality of sequences belonging to the same pathogen species, and so any sequence in the prior art that contains such a sequence anywhere along its length will be considered to read on this limitation. As for the “minimal” and “extended” splint sequences, these are not defined by the instant specification. Given that this oligonucleotide is a primer, and given how primers are generally used in the prior art, these splints will be considered to be the 3’ end before extension in an amplification reaction (for the minimal end splint) and after extension in the same reaction (for the extended splint), though see the 35 USC 112(b) Rejections above.
As this sequence involves nucleotides that are not present on the primer until after extension occurs (e.g. the extended 3’ end cDNA splint), and the claimed method only states that the listed oligonucleotides must be used, and not that they cannot be manipulated or must contain each listed component at the outset of the method, the listed component limitations will be considered to be met if they are taught in the prior art at any point during a library preparation process, and not just when an oligonucleotide is introduced. For example, if a primer does not initially contain a pathogen specific consensus sequence upon binding to a target, but does contain such a sequence after extension, then the primer will be considered to contain the pathogen specific consensus sequence as claimed.
For the 3’ and 5’ adapters, the term “adapter” is stated in para. 102 to generally refer to linear oligonucleotides which can be ligated to a nucleic acid molecule, but this is not a requirement, as later in the same paragraph it is noted that adapters may be attached via PCR. The “coupling sequence” for each adapter is not specified in the instant specification, but will be interpreted as the specific portion of the overall adapter that is used for attachment to a nucleic acid molecule. Thus, the “a 3’ adapter” and “a 5’ adapter” terms will be interpreted as the entirety of the adapter sequence, where the barcode and coupling sequences are contained within the adapters.
In claim 2:
For the pathogen specific template switching oligonucleotide, a “template switching motif” will be considered the portion of the oligonucleotide that renders it capable of being used for template switching. This will also apply to the “template switching motif” of the generic template switching oligonucleotide and the generic cDNA coupler reverse primer oligonucleotide.
For the “generic tailing motif” as recited in (vi) and (ix), no specific definition for tailing is given by the instant specification, and so this will be considered any non-complementary tail sequence present at either end of an oligonucleotide.
Generally in claim 2, the guidance provided in (c)(i) above is also followed here. For example, if in the prior art a pathogen specific template switching oligonucleotide exists that only contains a pathogen specific consensus sequence after extension, then the prior art will still be considered to read on the pathogen specific template switching oligonucleotide as claimed.
In (x), “a rDNA blocking duplex oligonucleotide” is stated. Such an oligonucleotide is not defined in the instant specification. However, in para. 100, it is stated “In some embodiments, the methods disclosed here comprise using a single DNA duplex matching homologous rRNA sequences from ITS genomic loci in mammalian species. In some embodiments, the single DNA duplex has 3' hexanediol-modified strands to block DNA polymerase processivity.” Such a description appears to describe a potential rDNA blocking duplex oligonucleotide, but is not considered limiting. Thus, the “rDNA blocking duplex oligonucleotide” is considered to generally encompass rDNA that can be used to block any function during library preparation, such as the blocking of hybridization or the blocking of oligonucleotide extension.
These interpretations apply to additional claims that may include these terms (e.g. claim 62).
It is also noted that claim 2 requires one or more of the listed oligonucleotides, as evidenced by the “and/or” between options (ix) and (x).
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2, 7, 9, 12, 14, 16, 21, 64-67, 69, and 70 are rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS), in view of Lee (Dissertation, 2020), and in view of Sims et al. (WO 2019/104337 A1).
Chappleboim teaches RNA-sequencing protocols for the detection of SARS-CoV-2 (Title and Abstract; instant claims 12 and 69). The basic workflow is shown in Figure 1, where mRNA capture on beads, reverse transcription and PCR, and then sequencing and subsequent analysis is done to detect particular target genes in sequence reads. Chappleboim utilizes sample-specific barcodes, because multiple samples may be pooled with their method (see Figure 1, Abstract, and “Barcoded RT primers added to lysed samples prime RT reactions” on page 4). Chappleboim utilized this method to analyze two targets in a multiplex setting – the N1 amplicon and the E amplicon (page 8, “Multiple Target Assay”). Figure 2D notes that these target sequences conform to reference genomes in 99.9% of reads, and so would qualify as “pathogen specific consensus sequences” under the claim interpretation described above (and also meet the limitations of instant claim 67, as this provides evidence that these target genes are in the viral genome). On page 11, Chappleboim utilizes this method on clinical samples that were both positive and negative for SARS-CoV-2 (“Robotic ApharSeq on clinical samples” and Figure 6). Page 14 shows how this method may be utilized to quickly, cheaply, and efficiently evaluate many samples with a nasopharyngeal swab, where samples are eventually sent to a sequencing site (Figure 7, and see page 2, para. 2 which specifically notes that nasopharyngeal swabs are used for SARS-CoV-2 testing (instant claim 7, also this testing is done on humans specifically, see Figure 1C, page 8, para. 3, Figure 5C, and Figure 7 for example, instant claims 14 and 70). On pages 16-18, Chappleboim describes their primer design. For both the RT-primers and the PCR-primers, target-specific portions were utilized (see the N1 and E portions of the primer design). It is noted that the reference also specifically states the method is “extraction-free” (see Title), but this refers to extraction from the sample lysis buffer in which the sample arrives (page 4, para. 1). The reference does later either isolate RNA from the rest of a sample via polyT capture (page 18, para. 4) or SPRI beads (page 18, para. 5). These methods are also listed on page 4, para. 1. However, under the interpretation of “extracting” described above in the “Claim Interpretation” section, the fact that viral and human RNA is directly accessible by capture beads upon their contact with one another indicates that this reference performs “extracting” as claimed.
However, the oligonucleotides of Chappleboim described in Figure 1 do not appear to meet all the limitations of the oligonucleotides of the instant claims.
Lee presents a new RNA sequencing technology while also providing methods for capturing, barcoding, reverse transcribing, and amplification (Abstract). Section 4.5 provides a molecular workflow for sequencing library preparation (starting on page 56). Section 4.5.2.1 and Figure 4.14 describe and show this workflow.
In section A, target mRNAs are captured using oligo-dT beads, where the oligo-dT binds to the mRNA, thus acting as a splint.
In sections B and C, target mRNAs are annealed to a PCR primer sequence comprising a PCR handle, a UMI, and an oligo-dT sequence. Reverse transcription is then performed to obtain cDNA. In section 4.5.3.1 on pages 59-60, it is noted that entirety of the UMI-dT sequence is composed of DNA. Under the interpretations provided in the 35 USC 112(b) Rejections section and the “Claim Interpretation” sections above, the dT portion (and the following two nucleotides) that initially binds to the mRNA can be considered analogous to the “minimal 3’ end cDNA splint,” and the cDNA portion that is then extended from this minimal portion is analogous to the “extended 3’end cDNA splint.”
In section H, sequencing adapters are added via PCR, where the adapters include complementary sequences to the fragments (the PCR handle and R2 in H, which act as coupling sequences). These adapters also include P5 and P7 sequences, respectively, as well as i5 and i7 sequences, which are index sequences (page 58, para. 1). As noted in para. 99 of the instant specification, the barcodes of the invention can be index sequences, and so these i5 and i7 sequences are analogous to the barcodes of the adapters. This workflow is then used with particular cell types on pages 61-65, but these are cancer cells, and pathogens are not described. It is noted that in this second example of Lee it is specified that the sequencing library is used to produce sequencing reads, where gene expression can be analyzed (see Figure 4.15 and section 4.6.3.7 on page 64).
However, this section of Lee does not specify a target mRNA sequence, and so it is unclear if the primer shown in B and C contains a pathogen specific consensus sequence. Additionally, the composition of the oligo-dT bead is not clear, and so it is not clear if this sequence has a UMI or a sample-specific barcode. Finally, the types of samples used with this workflow are not specified on pages 56-60.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to combine the methods of Chappleboim and Lee to arrive at the method of instant claim 1. Specifically, Lee teaches a method of creating a sequencing library from mRNA, and Chappleboim teaches a method of applying a sequencing library to effectively detect pathogens in samples. The methods of Lee involve mRNA released from single cells (see page 56, Section 4.5.2.1), and mRNA is the same starting material used by Chappleboim (e.g. Figure 1), thus demonstrating that the manipulations described by both experiments would work from the same starting sample containing target mRNA. Chappleboim teaches that their library preparation methods have low cross-contamination (page 3, para. 1), can analyze multiple genes (Figure 5), and are cheap and scalable for large amounts of samples (Figure 7), which are benefits that would be motivating to the ordinary artisan for detecting pathogens in a sample. In Chappleboim’s two-step PCR protocol, after reverse transcription, PCR is performed and then index primers are used (see pages 18-19). Thus, this version of their method corresponds well to steps A (bead capture), B-C (reverse transcription), E (initial PCR), and H (index adapter use) of Lee. Due to these similarities, it would be prima facie obvious to use the teachings regarding sequencing library oligonucleotide design of Lee in the method of Chappleboim to detect target SARS-Cov-2 genes. Because Chappleboim already teaches the use of these elements (with the UMI-annealing oligonucleotide of B of Lee being analogous to the RT-primer of Chappleboim), altering their design slightly to align with the guidance of Lee while still being used for the same purposes would provide a reasonable expectation of success.
Sims teaches methods for capturing single-cell lysates, including the use of dual barcode capture beads (Abstract). The provided barcodes can be used for inexpensive library preparation for RNA sequencing (paras. 2-3). Capture beads can contain oligonucleotides with oligo dT sequences, UMIs that are distinct for each oligonucleotide on the bead, a PCR handle, and a bead-specific barcode (para. 15). In para. 90, Sims notes that when cells and barcoded beads are placed in microwells, cell lysate can be generated, and mRNA can be pre-amplified. The resulting amplified RNA can then be reverse transcribed with primers that contain UMIs. The use of this second UMI is done so that “genes can be quantified from sequencing data based on the number of UMIs associated with each gene rather than the number of reads, mitigating noise and bias that result from exponential amplification by PCR.” Thus, Sims teaches UMIs on capture oligonucleotides and on reverse transcription primers.
Specifically regarding the oligo-dT bead, the teachings of Chappleboim already teach the use of a dT sequence attached to a bead (Figure 1A). Lee teaches a dt sequence and a 3’ splint in Figure 4.14. However, on page 9, Lee teaches the use of a barcoding primer bead, where said bead contains a handle, a cell barcode, a UMI sequence, and a dT sequence for tagging mRNA specifically (see Figure 2.2). The cell barcode is used to tag individual cells in a cell-specific method (see para. below the figure). This structure is very similar to the oligonucleotide on the capture beads described by Sims. In Sims, these bead oligonucleotides captured targets, where each capture oligonucleotide contained a cell barcode and a UMI, along with a dT sequence. In considering these references together, as all three are drawn to library construction, the ordinary artisan would be motivated to add the sample specific barcode (already present in the samples of Chappleboim, see the discussion of the RT primer below) and a UMI to the capture oligonucleotides on the beads of Chappleboim. In Chappleboim, samples are quickly hybridized to beads, and the RT primers are hybridized to the sequences. Then, samples are pooled, and reverse transcription and PCR occur in the pooled sample (see Figure 1 caption). By specifically tagging the beads with sample information, this allows for the identification of particular beads (and their associated hybridized targets) in the pooled sample, should they need to be recovered or more closely examined. In a similar manner, by adding UMIs to the capture oligonucleotides, if further analysis of the initial target nucleic acids sequences was desired, the capture oligonucleotide/target duplex could be removed from the beads and further analyzed. This then essentially tags each specific target molecule with a sample and unique tag. This would allow for granular analysis of the targets, and would aid in informing with regard to any particular mutation or strain information specific to a particular sample or molecule that is not captured by utilizing the target sequences on the RT and PCR primers. This would be useful in the event a patient is determined to be infected with SARS-CoV-2 by the general method of Chappleboim and the additional obtaining of strain/mutation information is vital, such as in the case of a more virulent strain that would require additional patient monitoring. There would be a reasonable expectation of success as Lee and Sims both teach that capture oligonucleotides can have UMIs, barcodes, and dT sequences.
Thus, Chappleboim, in view of Lee, and in view of Sims teaches oligonucleotide (b)(i).
Specifically regarding the pathogen specific oligonucleotide primer, Chappleboim teaches a reverse transcription primer with a PCR handle, a UMI, a sample barcode, and a target sequence (which would act as the pathogen specific consensus sequence). During reverse transcription, the minimal and extended cDNA splints would also naturally exist on this primer before and after extension, according to the guidance provided in the “Claim Interpretation” and 35 USC 112(b) Rejections above. Sims teaches that reverse transcription primers can contain UMIs that are distinct from UMIs that exist on capture beads, as all the UMIs would be unique (see paras. 15 and 90). It would be prima facie obvious that the RT primers would be designed such that this is the case, as the UMIs on the RT primers are serving an entirely different function than those on the capture oligonucleotides (i.e. labelling sequencing reads of amplicons rather than labelling beads/initial hybridized targets), and by making these sets of UMIs distinct, this would eliminate confusion during analysis. Thus, the creation of a second set of UMIs distinct from those on the capture beads would be prima facie obvious. There would be a reasonable expectation of success in adding this set of UMIs as Chappleboim teaches at least 10 nucleotides can be present in a UMI (page 16), and so the potential creation of a over a million distinct UMIs (410) is already possible in the reference.
Thus, Chappleboim, in view of Lee, and in view of Sims teaches oligonucleotide (b)(ii).
Specifically regarding the indexed adapters, as noted above, Lee teaches adapters with the claimed structure. These adapters are used with Illumina sequencing methods (see Figure 4.14). Chappleboim notes that separate primers can be provided that utilize sequencing adapters (see the top of page 18), and also teaches the use of Illumina sequencing (see page 5, para. 1, page 17, para. 1, and page 19, para. 2 for example). While the P5 and P7 sequences for the Illumina adapters are added during normal PCR in Chappleboim, this still leaves the index sequences and target complementary sequences as shown in Lee. Chappleboim teaches that their sequencing adapters contain index sequences (see the top of page 18), and so it would be prima facie obvious to use the remaining structure of the sequencing adapters shown in Lee (i.e. the i5 and i7 sequences and the sequences complementary to the amplification product) as the sequencing adapters in Chappleboim. The complementary sequences would allow for hybridization of the adapters and the amplification products, and as Lee notes in the caption of Figure 4.14, the adapter sequences as a whole are then added via additional PCR. Chappleboim also notes that these additional adapters are added via PCR, and not another method such as ligation (see the top of page 18). Therefore, the sequencing adapters of the references provide the same function (labeling the amplification products so they may be sequenced and more easily analyzed), and are added the same way. The use of the sequencing adapter designs of Lee in Chappleboim would therefore amount to simple substitution. MPEP 2143 I (B) states, “The rationale to support a conclusion that the claim would have been obvious is that the substitution of one known element for another yields predictable results to one of ordinary skill in the art.” As both adapter designs are known in the art, and their use is made clear by both Chappleboim and Lee, such substitution would be prima facie obvious.
Thus, Chappleboim, in view of Lee, and in view of Sims teaches oligonucleotides (b)(iii) and (b)(iv), and thus teaches each element of instant claim 1.
Therefore, Chappleboim, in view of Lee, and in view of Sims renders prima facie obvious claims 1, 7, 12, 14, 67, and 69-70.
Regarding claims 2 and 62, specifically regarding the pathogen specific enrichment coupler reverse primer, Chappleboim teaches that their PCR primers contain portions complementary to the target cDNA (which is a pathogen specific consensus sequence), and as they are primers, they have a minimal end at the outset, and extended ends once PCR extension begins. Chappleboim also notes that additional barcodes can be added via PCR primers (see Figure 1B and the “pool barcode”). Lee and Sims also teach that their PCR primers have a UMI. As Chappleboim teaches that the goal of their method is to be performed on a large scale with a great deal of pooling, it would be prima facie obvious to make this pooled barcode an additional UMI. This would ensure that no two pooled barcodes are the same, and would also provide a check on the method, improving accuracy (i.e. sequences that have successfully gone through each pre-sequencing step would have two UMIs, and so if a read was missing one of these sequences, it could indicate an error in the method). As UMIs are known in the art, as evidenced by Chappleboim, Lee, and Sims, and only the barcode of the reverse primer shown in Figure 1B would be altered with said UMI, there would be a reasonable expectation of success.
Specifically regarding the generic cDNA coupler reverse primer oligonucleotide, Lee teaches that during their library preparation methods, 2nd strand cDNA synthesis involves a template-switching oligonucleotide with a template switching motif (the rG motif) and a tailing motif (the TS oligo). In Figure 1, Chappleboim does not explicitly show 2nd strand cDNA synthesis. However, the resulting cDNA strands contain both the forward and reverse primer sequences (see Figure 1), thus providing evidence that 2nd cDNA strands may be generated during the PCR of Chappleboim. As Lee provides a way of generating a 2nd cDNA strand before PCR begins through the simple use of a template-switching oligonucleotide, it would be prima facie obvious to use such an oligonucleotide in the method of Chappleboim, in view of Lee, and in view of Sims. This is because such synthesis would allow for both the forward and reverse PCR primers to both begin amplifying targets immediately upon the first cycle, thereby generating more amplification product. This would provide more product for sequencing and thereby increase the odds that SARS-CoV-2 would be successfully detected in the sample. As Lee teaches template-switching in the context of sequencing library preparation, there would be a reasonable expectation of success.
Regarding claims 9 and 66, as the samples of Chappleboim are from human nasopharyngeal swabs, and as shown in Figure 1C, the sequencing analysis notes that both human and viral DNA may be present in the samples. As the instant specification does not define a “plurality,” the presence of nucleic acids from two organisms (human and SARS-CoV-2) will be considered a plurality.
Regarding claim 16, Figure 7 of Chappleboim shows the desired application of their method – where many samples are collected in arrays, processed simultaneously, additionally pooled, and then sequenced. The reference states that this can be used with initially pooled samples (page 13, para. 6). Thus, this reference teaches that multiple pooled samples on plates (where each sample contains RNA from multiple subjects) can be processed and sequenced simultaneously.
Regarding claim 21, the purpose of the method of Chappleboim is to detect SARS-CoV-2 infection (Abstract). Throughout the reference, Chappleboim teaches that their assay can be used to detect viral loads (Figures 4-5), and clinical samples were specifically tested for viral molecules (with the data shown in Figure 6). Thus, it would be prima facie obvious to the ordinary artisan that the method of Chappleboim could be used to determine infection status based on the presence or absence of targeted viral genes in the analysis of the sequencing library.
Regarding claim 64, as Chappleboim, in view of Lee, and in view of Sims teaches the analysis of RNA, their nucleic acid sample would naturally comprise RNA.
Regarding claim 65, Chappleboim teaches that their detection methods involved the use of clinical samples (Abstract, page 11, Figure 6, and page 19, para. 4).
Claims 17 and 72 are rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS), in view of Lee (Dissertation, 2020), in view of Sims et al. (WO 2019/104337 A1), and further in view of Takara (2019).
Chappleboim, in view of Lee, and in view of Sims teaches the methods of claims 1-2, 7, 9, 12, 14, 16, 21, 64-67, 69, and 70, as described above. Chappleboim generally teaches that their library preparation method is a multi-tube method – see pages 18-19, “Option 1: Purification and hybridization on PolyT beads” and “cDNA synthesis and Library preparation.” However, neither Chappleboim nor Lee teaches the use of PCR pre-amplification. Sims teaches pre-amplification, but this comes before reverse transcription is performed (e.g. paras. 28, 88, and 102).
Takara teaches a particular master mix for performing PCR pre-amplification. The main benefit of said pre-amplification is stated to be being able to use low amounts of starting nucleic acids, and said pre-amplification can be used with cDNA (page 1, “Introduction”). Pre-amplification provides lower Ct values, meaning amplification itself would be more efficient, and can be successful with as little as 100 pg of starting material (Figures 1 and 2). Takara also teaches that their mix is highly unbiased (Table I).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Takara to add a PCR-preamplification step to the method of Chappleboim, in view of Lee, and in view of Sims. Such a step would occur after reverse transcription, as the pre-amplification can effectively be used on cDNA. Takara teaches a commercially available kit for performing said pre-amplification, as well as many benefits associated with including such a step in amplification methods. The ordinary artisan would be motivated to specifically use pre-amplification in the context of Chappleboim, in view of Lee, and in view of Sims because this would increase the detection rate of SARS-CoV-2 in a sample, even if the virus is present in a sample in very low amounts, which can lead to more accurate diagnostics and earlier patient intervention, potentially improving patient outcomes, particularly for patients who are at increased risk of disease complications. There would be a reasonable expectation of success because the resources and protocols for using such pre-amplification are readily available from Takara.
Thus, claims 17 and 72 are prima facie obvious over Chappleboim, in view of Lee, in view of Sims, and further in view of Takara.
Claim 63 is rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS), in view of Lee (Dissertation, 2020), in view of Sims et al. (WO 2019/104337 A1), and further in view of Liu et al. (Front. Microbiol., 2019).
Chappleboim, in view of Lee, and in view of Sims teaches the methods of claims 1-2, 7, 9, 12, 14, 16, 21, 64-67, 69, and 70, as described above. However, none of these references teach the use of a rDNA blocking oligonucleotide.
Liu focuses on analysis of gut microorganisms in marine animals, and specifically, how to effectively analyze said microorganisms in the presence of host DNA (Abstract). Liu focused on the 18S region of the microorganisms in question, and developed a blocking primer to “block the amplification of host DNA in a complex sample for the detection of eukaryotic species of interest,” as the blocking primer preferentially binds to host DNA (pages 2, columns 1-2 joining para.). The blocking primer was based on rDNA regions that corresponded to rRNA in the microorganisms (particularly eukaryotic bacteria; page 2, column 2, para. 3). Multiple blocking primers were developed (page 3, column 1, para. 4). These blocking primers were then used in methods involving amplification, library preparation, and sequencing (pages 3-4, “Amplification With Blocking Primers” and “Library Construction and Sequencing”). Liu teaches that the blocking primers were able to successfully suppress unwanted amplification of host sequences during PCR (page 10, columns 1-2 joining para.).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the guidance provided by Liu in the context of Chappleboim, in view of Lee, and in view of Sims. Specifically, Chappleboim, in view of Lee, and in view of Sims deals with examining viruses from human nasopharyngeal samples, which, by nature of the sampling, would likely include human, bacterial, and viral nucleic acids. Liu teaches means by which to block undesired sequences from being amplified utilizing rDNA blocking primers. While Chappleboim does analyze host target genes in some of their embodiments (e.g. Figure 1C and Figure 5D) this is not a requirement for every embodiment (e.g. Figure 5B). Additionally, in determining if a particular patient sample has SARS-CoV-2, from a diagnostic perspective, it would not be necessary to also analyze the human DNA in a sample as well. In fact, the presence and amplification of such DNA may hinder virus amplification and detection by providing false or noisy results. And although the PCR primers used in Chappleboim, in view of Lee, and in view of Sims do contain target specific portions, they also contain barcodes/UMIs/PCR handles that would allow for potential non-specific or non-desired amplification. As Liu teaches that utilizing rDNA primers specifically can block human amplification without interfering with PCR primer function, and Liu carefully lays out design guidance for creating an effective blocking primer (see the guidance at the top of page 3 of Liu, for instance), the design and inclusion of such blocking primers would not require a great deal of time and resources to include in Chappleboim, in view of Lee, and in view of Sims, and their inclusion would potentially improve the analysis efficiency of sequencing results. Furthermore, as human DNA is double-stranded, it would be prima facie obvious to create duplex rDNA blocking primers to target potentially more interfering human sequences. There would be a reasonable expectation of success in the design and inclusion of the rDNA sequences as Liu teaches they can explicitly be used with amplification and library creation.
Thus, claim 63 is prima facie obvious over Chappleboim, in view of Lee, in view of Sims and further in view of Liu.
Claim 68 is rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS), in view of Lee (Dissertation, 2020), in view of Sims et al. (WO 2019/104337 A1), and further in view of Kim et al. (Cell, 2020).
Chappleboim, in view of Lee, and in view of Sims teaches the methods of claims 1-2, 7, 9, 12, 14, 16, 21, 64-67, 69, and 70, as described above. As noted above, Chappleboim teaches the use of pathogen specific consensus sequences (the N1 amplicon and the E amplicon), but these are not taught to be transcription-regulation sequence motifs.
Kim teaches the use of DNA and RNA sequencing to create a high-resolution map of the SARS-CoV-2 transcriptome (Summary). The reference teaches that coronaviral RNA contains motifs called transcription-regulatory sequences (TRSs) that are immediately adjacent to open reading frames (ORFs; page 914, column 2, para. 2 and Figure 1), and Kim was able to map TRSs (page 916, column 1, para. 1 and Figure 2A). The examination of these sequences allowed for the determination of fusion and discontinuous transcription sites (Figure 3D-G), and it was noted that TRSs can affect sgRNA production (page 917, columns 1-2 joining para., for example).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the teachings of Kim to examine additional SARS-CoV-2 genes with the method of Chappleboim, in view of Lee, and in view of Sims, and in particular to analyze TRSs. As TRSs are important for viral transcription, the ordinary artisan would recognize that they would be valuable to study, particularly as their manipulation may lead to changes in sgRNA production or transcription that would render a virus weaker or non-functional, which would have implications for disease control and treatment. Though Chappleboim, in view of Lee, and in view of Sims is focused on diagnostics, the ordinary artisan would also recognize the general utility in using Chappleboim, in view of Lee, and in view of Sims as a sequencing method to analyze particular viral variants in individual patients, and targeting TRSs could provide information about disease prognosis for particular viral strains, as TSRs are vital to viral function. As Kim teaches that they were capable of sequencing TRSs with RNA sequencing, and specifically utilizes reverse transcription and PCR methods (see e3, para. 1), the ordinary artisan would have a reasonable expectation of success in designing primers in the method of Chappleboim, in view of Lee, and in view of Sims capable of targeting TRSs. In particular, this would only involve altering the existing primer structure of Chappleboim, in view of Lee, and in view of Sims to contain TRS-specific sequences in the target region of the primer – the other features of the primers, such as UMIs, would remain the same. As Kim teaches the discovery of TRS region sequences, the ordinary artisan would be capable of then incorporating those sequences (or their complements) into a primer.
Thus, claim 68 is prima facie obvious over Chappleboim, in view of Lee, in view of Sims, and further in view of Kim.
Claim 71 is rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS), in view of Lee (Dissertation, 2020), in view of Sims et al. (WO 2019/104337 A1), and further in view of Bibillo et al. (US 2019/0071711 A1).
Chappleboim, in view of Lee, and in view of Sims teaches the methods of claims 1-2, 7, 9, 12, 14, 16, 21, 64-67, 69, and 70, as described above. Chappleboim generally teaches that their library preparation method is a multi-tube method – see pages 18-19, “Option 1: Purification and hybridization on PolyT beads” and “cDNA synthesis and Library preparation.” None of these references teach the use of single-pot, open-tube chemistry for library preparation.
Bibillo teaches methods for amplifying and analyzing nucleic acid samples, particularly for creating cDNA libraries (Abstract). Specifically, the reference teaches various methods for single-pot library preparation (e.g. paras. 72, 74-75, 165, 207, and 212). In paras. 338-339, Example 4 shows that an RNA molecule was captured, reverse transcribed, and amplified in an open tube reaction. Para. 341 and Example 5 show a similar single-pot reaction that is specifically used for library preparation. Bibillo also specifically states that their methods are compatible with capture on a solid support, such as a bead (para. 224). Paras. 53-54 note advantages of their invention, including quick library preparation (which is noted to be useful in clinical settings), efficiency and simplicity, and high quality library preparation.
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the guidance provided by Bibillo in the method of Chappleboim, in view of Lee, and in view of Sims to arrive at the method of claim 71. Specifically, Bibillo teaches that library preparation, with reverse transcription and amplification, can occur in a single tube, and also teaches that initial RNA capture methods can be added to this. In Chappleboim, in view of Lee, and in view of Sims, RNA capture, reverse transcription, and amplification are performed, followed by sequencing. Though Chappleboim notes that samples can be pooled, the purpose of the invention generally is to rapidly detect SARS-CoV-2. Thus, the ordinary artisan would recognize the value in preparing a single sample in a single reaction tube, as this can all be performed in a clinical setting without needing to ship the sample to another location, saving time, thereby more quickly providing accurate and important information to patients. Such benefits are also explicitly stated by Bibillo. There would be a reasonable expectation of success because the same general library preparation steps are taught by Bibillo and Chappleboim, in view of Lee, and in view of Sims.
Thus, claim 71 is prima facie obvious in view of Chappleboim, in view of Lee, in view of Sims, and further in view of Bibillo.
Claim 73 is rejected under 35 U.S.C. 103 as being unpatentable over Chappleboim et al. (medRxiv, 2020; cited in Applicant’s IDS), in view of Lee (Dissertation, 2020), in view of Sims et al. (WO 2019/104337 A1), and further in view of Parker et al. (Journal of Applied Microbiology, 2010).
Chappleboim, in view of Lee, and in view of Sims teaches the methods of claims 1-2, 7, 9, 12, 14, 16, 21, 64-67, 69, and 70, as described above. Chappleboim generally teaches that their library preparation method is a multi-tube method – see pages 18-19, “Option 1: Purification and hybridization on PolyT beads” and “cDNA synthesis and Library preparation. However, neither Chappleboim, nor Lee, nor Sims teaches the use of MDA pre-amplification.
Parker teaches amplification of viral RNA from environmental samples (Abstract). The goal of the reference was to perform whole transcriptome amplification (WTA) via an initial round of MDA followed by PCR, followed by targeted qPCR (page 216, “Introduction” and page 217, “WTA reactions” and “qRT-PCR”). Generally, the reference teaches that this process allows for successful amplification of environmentally relevant amounts of RNA, and thus small amounts of pathogens (page 221, “TransPlex WTA for small quantities of pathogens”).
Prior to the effective filing date of the claimed invention, it would have been prima facie obvious for one of ordinary skill in the art to use the guidance provided by Parker to add a WTA MDA-preamplification step to the method of Chappleboim, in view of Lee, and in view of Sims. Parker teaches this pre-amplification is compatible with downstream targeted PCR methods, and also teaches that this method can allow for detection of small quantities of viruses in samples. The ordinary artisan would thus be motivated to specifically use this pre-amplification in the context of Chappleboim, in view of Lee, and in view of Sims because this would increase the detection rate of SARS-CoV-2 in a sample, even if the virus is present in very low amounts, which can lead to more accurate diagnostics and earlier patient intervention, potentially improving patient outcomes, particularly for patients who are at increased risk of disease complications. There would be a reasonable expectation of success because the WTA method taught by Parker is commercially available, and so the resources and protocols for using such pre-amplification are readily available.
Thus, claim 73 is prima facie obvious over Chappleboim, in view of Lee, in view of Sims, and further in view of Parker.
Conclusion
No claims are currently allowable.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/F.F.G./Examiner, Art Unit 1681
/ANGELA M. BERTAGNA/Primary Examiner, Art Unit 1681
